The telomeric protein TRF2 is critical for the protection

MOL#39081

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The telomeric protein TRF2 is critical for the protection of A549

cells from both telomere erosion and DNA double-strand breaks

driven by salvicine

Yong-Wei Zhang, Zhi-Xiang Zhang, Ze-Hong Miao, Jian Ding

Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai

201203, People’s Republic of China

Molecular Pharmacology Fast Forward. Published on November 19, 2007 as doi:10.1124/mol.107.039081

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on November 19, 2007 as DOI: 10.1124/mol.107.039081

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Running title: TRF2 protects A549 cells from telomeric erosion and DSBs

Address correspondence to: Jian Ding, Ph.D. & M.D., Division of Anti-Tumor

Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China.

Tel: +86-21-58506600 ext 2423.

Fax: + 86-21-50806722. E-mail:[email protected]

The number of text pages: 19

Number of tables: 0

Number of figures: 5

Number of references: 39

Number of words in the Abstract: 221

Number of words in the Introduction: 498

Number of words in the Discussion: 913

Abbreviations: TRF2, telomere repeat binding factor 2; SAL, salvicine; ATM,

Ataxia-telangiectasia-mutated; ATR, ATM- and Rad3- related; DSB, DNA

double-strand break; Top2, topoisomerase II; ROS, reactive oxygen species; ChIP,

chromatin immunoprecipitation; SRB, sulforhodamine B; siRNA, small interfering

RNA; SSB, single-strand break.


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Abstract

Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be

involved in DNA damage response and telomere maintenance. Our previous report

found that salvicine (SAL), a novel topoisomerase II poison, elicited DNA

double-strand breaks and telomere erosion in separate experimental systems.

However, it remains to be clarified whether they share a common response to these

two events and in particular and if TRF2 involves in this process. In this study, we

found that SAL concurrently induced DNA double-strand breaks, telomeric DNA

damage and telomere erosion in lung carcinoma A549 cells. Unexpectedly, SAL led

to disruption of TRF2, independently of either its transcription or

proteasome-mediated degradation. By over-expressing the full-length trf2 gene and

transfecting TRF2 small interfering RNAs, we showed that TRF2 protein protected

both telomeric and genomic DNA from the SAL-elicited events. Notably, though both

the Ataxia-telangiectasia-mutated (ATM) and the ATM- and Rad3-related (ATR)

kinases responded to the SAL-induced DNA damages, only ATR was essential for the

telomere erosion. The study also showed that the activated ATR augmented the

SAL-triggered TRF2 disruption while TRF2 reduction in turn enhanced ATR function.

All these findings suggest the emerging significance of TRF2 protecting both

telomeric DNA and genomic DNA on one hand, and reveal the mutual modulation

between ATR and TRF2 in sensing DNA damage signaling during cancer

development on the other hand.


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Introduction

Telomere is the specialized ribonucleoprotein structure at the ends of

chromosomes in eukaryotic cells, which specifically protects chromosomes from end

fusion and genomic instability (Greider and Blackburn, 1996). Thus, telomere

integrity in cells thus plays an essential role in the control of genomic stability.

Dysfunction of telomeres leads to cell cycle arrest and initiates cell apoptosis or cell

senescence (Farazi et al., 2006; Herbig and Sedivy, 2006; Lechel et al., 2005).

Therefore, an increasing numbers of studies have focused on telomere maintenance.

Telomere maintenance depends heavily on telomere binding proteins, of which

telomere repeat binding factor 2 (TRF2) is a critical member. TRF2 is one of six

proteins consisting of the protein complex shelterin (de Lange, 2005). Shelterin has

been shown to be located on telomeres and to be related to telomere length regulation

and telomere structure maintenance (de Lange, 2005). However, recent reports have

revealed that TRF2 is recruited to DNA double-strand break (DSB) sites

immediatedly after DSBs take place (Bradshaw et al., 2005). The TRF2 protein is

phosphorylated in response to DSB signals (Tanaka et al., 2005). Loss or mutation of

TRF2 leads not only to telomeric structure destruction but also to DNA damage,

apoptosis or senescence concomitantly (Lechel et al., 2005; Smogorzewska and de

Lange, 2002; van Steensel et al., 1998; Wang et al., 2004). In addition, TRF2

promotes the polymerization activity of DNA polymerase β (pol β) on telomeric and

non-telomeric DNA substrates (Muftuoglu et al., 2006). As such, TRF2 is emerging


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as a potential target in cancer therapy.

Salvicine (SAL) is a novel diterpenoid quinone compound synthesized by the

structural modification of a natural product isolated from the Chinese medicinal herb

salvia prionitis lance (Zhang et al., 1999). It is now undergoing its phase II clinical

trials. SAL possesses potent in vitro and in vivo activities against malignant tumor cells

(Qing et al., 1999) functioning as a noninteractive topoisomerase II (Top2) poison (Lu

et al., 2005a; Lu et al., 2005b; Meng et al., 2001a). Moreover, SAL stimulates

intracellular reactive oxygen species (ROS), which subsequently elicits DNA DSBs

closely related with cell death (Lu et al., 2005b). Furthermore, SAL inhibits telomerase

activity and shortens telomere length in a telomerase-independent manner (Liu et al.,

2002; Liu et al., 2004). Due to the notion that the telomere erosion is critical for DNA

DSBs, we have investigated whether tumor cells share the common response to

telomeric and non-telomeric DNA damage upon the exposure to salvicine. In particular,

as TRF2 is increasingly recognized to be capable of functionally sensing DNA damage,

we further investigated if TRF2 is involved in salvicine-triggered telomere erosion and

DNA DSBs.

The aim of the present study is to unravel the effect of SAL on genomic and

telomeric DNA in a single system, to characterize the role of SAL on TRF2

expression, and to explore the possible involvement of TRF2 protein in SAL-driven

events. This will hopefully further our understanding of the distinct or unidentified

roles of TRF2 in cancer development.


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Materials and Methods

Drugs and chemicals. SAL was provided by the Department of Phytochemistry

in the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences.

The purity of the compound was more than 99%. VP16 and caffeine were purchased

from the Sigma Chemical Co. ( St. Louis, USA). MG-132 was the product of the

Biomol Biotech. Inc. (Plymouth Meeting, USA). Both SAL and VP16 were prepared

at 100 mM in DMSO. Caffeine was dissolved at 80 mM in distilled water and then

filtrated with 0.22 µm filter (Millipore, USA). MG-132 was dissolved at 20 mM in

DMSO. The aliquots of SAL, VP16 and caffeine stock solutions were stored at –20°C,

and MG-132 stock solution was kept in –80°C avoiding light. All drugs were diluted

to desired concentrations in full medium immediately prior to each experiment. The

final DMSO concentrations did not exceed 0.1%.

Cell culture. A549 cells were obtained from the American Type Culture

Collection (ATCC, Manassas, USA) and cultured in Ham's F12 medium (GIBCO

BRL, Grand Island, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine

serum (GIBCO BRL, Grand Island, USA), 100 µg/mL of streptomycin, 100 units/mL

of penicillin and 2 mM L-glutamine in a humidified atmosphere (5% CO2) at 37 °C.

Comet assays. DNA DSBs were evaluated using the neutral single cell gel

electrophoresis (neutral comet assays) as previously described (Olive et al., 1990)

with minor modifications (Lu et al., 2005a). Quantification was performed by

analyzing at least 50 randomly- selected comets per slide with the Komet 5.5 software


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(Kinetic Imaging Ltd., Nottingham, UK). The Olive tail moment (OTM) was selected

as the parameter that best reflects the degree of DNA damage (arbitrary units, defined

as the product of the percentage of DNA in the tail multiplied by the tail length), and

the data were expressed as mean ± SD.

Relative telomere length assays. Relative telomere length was detected by flow

cytometry (FLOW-FISH) via fluorescence in situ hybridization with a

fluorescein-conjugated PNA probe using the Telomere PNA Kit/FITC for Flow

Cytometry (DAKO, Glostrup, Denmark) according to the manufacturer’s protocol with

a slight modification (Liu et al., 2004). Relative telomere length was the ratio of the

telomere fluorescence intensity in treated cells to that in the control; the telomere

fluorescence intensity = (Mean FL1 with probe – mean FL1 without probe). The

fluorescence intensity of the control cells was taken as 100%.

Telomere FISH and TUNEL assays. Cellular telomere signals and DNA break

signals were detected with the Cy3-labeled telomere PNA probe and the FITC-labled

dUTP respectively. Using the Telomere PNA FISH kit (DAKO, Glostrup, Denmark),

telomere signals were performed first. Cells were cultured and treated on chamber

slides in 6-well plates and then fixed in 4% paraformaldehyde in PBS for 15 min.

Following washing with PBS, the cells were permeabilized in 0.1% TritonX-100 for 10

min and washed again with PBS. The slides were immersed in the pre-hybridization

solution for 10 min and subsequently in cold ethanol series (70%, 85% and 95%) and

left in air until dried. Fifty µL TUNEL reaction mixture containing the FITC-labeled


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dUTP and terminal deoxynucleotidyl transferase (TdT) (Roche, Switzerland) was

added to the slides. After incubation at 37°C for 1 h, the slides were washed three times

with PBS, air-dried and stained with 0.5 µg/mL DAPI for 30 min in the dark. Images

were taken using a Leica TCS Confocal Microscope (Leica, Deerfield, USA).

Western blot analyses. Protein levels were measured by Western blotting with

corresponding specific primary antibodies, including those against TRF2 (Imgenex,

USA), phosphorylated-H2AX (γ-H2AX) (Cell signal Biotech., USA),

Ataxia-telangiectasia-mutated kinase (ATM) (Rockland Inc., USA), ATM- and

Rad3-related (ATR) (Calbiochem, USA) and Myc (Cell signal Biotech., USA). Shown

were the representative data from separate experiments.

Immunofluorescence assays. A549 cells plated on coverslips in 24-well plates

were used to study the subcellular localization of TRF2 and γ-H2AX. Cells were

air-dried, fixed for 30 min with 4% paraformaldehyde in PBS (pH 7.4), and washed

twice with PBS. Then, the cells were incubated for 15 min with 0.2% TritonX-100 and

washed with PBS. After that, the cells were incubated in blocking buffer [3% bovine

serum albumin (BSA) in PBS] for 30 min before being incubated for 1 h with the

primary antibodies against TRF2 (Imgenex, USA) and γ-H2AX (Cell Signal, USA).

Following three washes (2 min each) with PBS, the cells were incubated for an

additional 1 h with the Alexa Fluor® 488-conjugated secondary antibody (Alexa Fluor®

488 goat anti-mouse IgG, Molecular Probes, USA). The cells were washed three times

and stained with 0.5 µg/mL DAPI for 30 min in the dark. Images were taken using a


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Leica TCS Confocal Microscope (Leica, Deerfield, USA). Fluorescence was

semi-quantitated by Photoshop 7.0 software.

RNA extraction and RT-PCR. Total cellular RNA was isolated from A549 cells

with the RNeasy Mini Kit (Qiagen, German) using the standard protocol for animal

cells. The semi-quantitative reverse transcription-PCR (RT-PCR) for trf2 mRNA was

performed with the QIAGEN OneStep RT-PCR Kit. Trf2 primers used were 5’-TCC

CAA AGT ACC CAA AGG C-3’(sense) and 5’-ACT CCA GCC TTG ACC CAC

TC-3’(antisense). GAPDH was used as the loading control with the primers of 5’-TCA

CCA TCT TCC AGG AGC GAG A-3’ (sense) and 5’-GCA GGA GGC ATT GCT

GAT GAT C-3’ (antisense). The Onestep RT-PCR program was run as the following:

for the reverse transcription, 50°C for 30 min and 95°C for 15 min; and for the PCR,

hot start 94°C for 2 min followed by 36 cycles of 94°C for 30 s, 60°C for 30 s and 72°C

for 45 s, and then 72°C, 10 min. The PCR products were resolved by 1.5% agarose

electrophoresis.

Telomere-ChIP. ChIP was carried out using a Chromatin IP (ChIP) assay kit,

following the manufacturer’s instructions (Upstate, USA). Cells were fixed with

formaldehyde for 10 min and then sonicated. Sonicated samples were pre-cleared and

precipitated with the TRF2 antibody (Imgenex, USA). The resulting precipitates (ppt)

were blotted onto a Hybond-N membrane (Amersham Biosciences, USA) under

vacuum. Aliquots of the supernatants before immunoprecipitation were taken out as

inputs. Telomere repeat sequences were detected with a TeloTAGGG telomere length


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assay kit (Roche Diagnostics, Switzerland). Briefly, the membrane was hybridized

with a DIG-labeled telomere probe. The hybridized telomeric DNA was probed with

the anti-DIG antibody. Telomere signals were semi-quantitated with InGenius

Bio-Imaging System (Syngene, Cambridge, UK). Telomeric DNA in CHIP (%) =

Telomeric DNA signal of ppt / Telomeric DNA signal of input × 100%.

Construction of recombinant expression plasmids : pCDNA3.1-myc-his(-)A-

trf2 and pCDNA3.1-myc-his(-)A-trf2∆B∆M. pBabe puro-trf2 and pBabe

puro-trf2∆B∆M were taken as the template plasmids, which were kind gifts from

Professor de Lange Titia in the Rockfeller University. The full-length trf2 gene was

amplified by PCR with two primers carrying EcoR I and BamH I sites: 5’-GTG CTG

GAT ATC TGC AGA ATT CCA ATG GCG GGA GGA GGC GGG AGT AGC G-3’

(sense) and 5’-CTT GGT ACC GAG CTC GGA TCC GTT CAT GCC AAG TCT

TTT CAT GGT CCG CC-3’ (antisense). The trf2∆B∆M PCR primers with EcoR I and

BamH I sites used for amplification were 5’-GTG CTG GAT ATC TGC AGA ATT

CCA ATG GAG GCA CGG CTG GAA GAG GCA GTC A-3’ (sense) and 5’-CTT

GGT ACC GAG CTC GGA TCC TTC TAC AGT CCA CTT CTG CTT TTT TGT

TA-3’ (antisense). Eighty ng template plasmids were added to a 50 µL PCR system,

including 1×buffer [10 mM Tris-HCl (pH8.3), 1.5 mM MgCl2, 50 mM KCl ], 50 µM

dNTPs, 3 units of Taq DNA polymerase, and 0.1 µM of specific primers. The PCR

mixture was subjected to 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min.

The amplified products were digested with EcoR I and BamH I and ligated to the


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corresponding cloning sites in the pcDNA3.1/myc-His(-)A mammalian expression

vector (Invitrogen, USA).

Transfection of plasmids and selection for stable expression cell lines. The

plasmids were transfected with Lipofectamine 2000 (Invitrogen, USA). For each

transfection, 4 µL lipofectamine 2000 was added to 200 µL opti-MEM and incubated

at room temperature for 5 min. The mixture was then added to further 200 µL

opti-MEM with 2 µg plasmid DNA for 20 min. The transfection complexes were then

applied to cells within 600 µL opti-MEM medium. After 4 h incubation, 1 mL fresh

F12 medium was added to the cells. Twenty-four hours later, the medium was

replaced with fresh full medium. Subsequently, positive transefected cells were

selected with 500 µg/mL G418. The Myc tag was detected by Western blotting.

Small interfering RNA (siRNA) and transfection. Three pairs of TRF2

siRNA sequences were designed as #1: 5'-CCU UCU UUA GUG GUU UGC UUA

UdTdT-3' (sense) and 5'-AUA AGC AAA CCA CUA AAG AAG GdTdT-3'

(antisense); #2: 5'-ACC UAA UUU CAU UCU UUG UGA AdTdT-3’ (sense) and 5'-

UUC ACA AAG AAU GAA AUU AGG UdTdT-3’ (antisense); #3: 5'-CGG CUU

UCA UUU CCA CAG AAU UdTdT-3' (sense) and 5'-AAU UCU GUG GAA AUG

AAA GCC GdTdT-3' (antisense). Their target sequences are all located within the

3’-UTR region of the TRF2 cDNA( #1: nts.2199-2220; #2: nts. 2702-2723; #3: nts.

1633-1653). ATR siRNA sequences were 5’-CCU CCG UGA UGU UGC UUG

AdTdT-3’ (sense) and 5’-UCA AGC AAC AUC ACG GAG GdTdT-3’ (antisense)


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(Casper et al., 2002). ATM siRNA sequences were 5’-CAU ACU ACU CAA AGA

CAU UdTdT-3’ (sense) and 5’-AAU GUC UUU GAG UAG UAU GdTdT-3’

(antisense) (Nur et al., 2003). All the RNA oligonucleotides and the mock siRNAs

were synthesized by the Genepharma Co. (Shanghai, CHINA). siRNAs were

transfected with the Oligofectamine reagent (Invitrogen, USA) according to the

manufacturer’s instructions.

Sulforhodamine B (SRB) assays. The sensitivity of A549 cells to SAL was

evaluated by SRB assays (Lu et al., 2005a). The cell proliferation inhibition rate was

calculated as: proliferation inhibition (%) = [1-(A515 treated/A515 control)]×100%.

In vitro assays for ATR kinase activity. ATR kinase assays with immuno-

precipitated ATR from A549 cells were carried out as described previously (Canman

et al., 1998) with minor modifications. After treatment, cells were trypsinized and

washed three times in cold PBS and the pellets were lysed for 10 min at 4°C in 200

µL lysis buffer [25 mM HEPES (PH 7.4), 300 mM NaCl�5 mM EGTA, 1 mM MgCl2,

0.5% NP-40, 10 µg/mL aprotinin, 1 µg/mL pepstatin A, 10 µg/mL leupeptin, 2 mM

PMSF, 20 nM microcystin-LR, 25 mM NaF]. Pre-cleared lysates were incubated with

the ATR primary antibody (Calbiochem, USA) and protein A/G agarose at 4°C

overnight. The complexes were washed three times with lysis buffer and then twice

with kinase reaction buffer [50 mM HEPES (pH 8.0), 10 mM MnCl2, 2.5 mM EDTA,

1 mM DTT]. The beads were then re-suspended in 30 µL of kinase reaction buffer

containing 10 mM ATP and 5 µg p53 recombinant protein (Lab Vision Corporation,


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Fremont, USA). The mixture was incubated at 30°C for 30 min. Then 30 µL of

2×SDS loading buffer was added and the phosphorylated proteins were separated by

SDS−PAGE, then proceeding for normal Western blotting with the

anti-phospho-p53(Ser15) antibody (Cell signal Biotech., USA). Bands of

phospho-p53 were scanned and semi-quantitated with the InGenius Bio-Imaging

System (Syngene, Cambridge, UK). The activity of ATR in the mock-siRNA-alone

group was taken as 100%.

Statistical analyses. All the data are represented as mean values ± SD. The

significance of differences between means was assessed by Student’s t test, with

P<0.05 and P<0.01 considered statistically significant.


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Results

SAL induces both DNA DSBs and telomeric erosion in A549 cells. To detect

whether SAL concurrently induces both DNA DSBs and telomeric erosion, we

treated A549 cells with different concentrations of SAL for 3 h. Using Comet assays,

we found that SAL elicited DNA DSBs as shown by “comet tails” in neutral

electrophoresis (Fig. 1A). The degree of DNA DSBs was augmented as the

concentrations of SAL increased (Fig. 1A). The increase of the γ-H2AX levels and

the formation of γ-H2AX foci in the SAL-treated A549 cells further confirmed the

result from Comet assays (Fig. 1B). These data indicate that SAL produces genomic

DSBs and activates a DNA damage response in A549 cells.

At the same time, SAL also damaged and shortened telomeric DNA. Using the

telomere-specific probe and dUTP-FITC to label telomeric DNA and DNA breaks,

respectively, their co-localization was defined as DNA break foci at telomere sites

(Bradshaw et al., 2005; Karlseder et al., 2004). From Fig. 1C, the co-localization of

DNA break signals and telomere signals appeared in the cells treated with 25 µM

SAL. With increasing concentrations of SAL, DNA breaks were observed across the

nuclei in a diffuse pattern and DNA damage at telomere sites increased concurrently.

Moreover, as the concentrations of SAL increased from 25 µM to 100 µM, the

relative telomere length reduced progressively from 87.7 ± 6.6 % to 53 ± 1.5 % (Fig.

1D). The data above demonstrate that SAL can induce both telomeric and

non-telomeric DNA damage in A549 cells.


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SAL disrupts TRF2 protein in A549 cells. TRF2 protein has been shown to bind

to telomeric DNA and protects the structure of telomeres (Stansel et al., 2001;

Yoshimura et al., 2004). Release of TRF2 from telomeres would leave the telomeric

DNA unprotected (Li et al., 2005). In addition, TRF2 has also been suggested to be

related to non-telomeric DNA damage and DNA repair events (Bradshaw et al., 2005;

Muftuoglu et al., 2006; Tanaka et al., 2005). Using immunofluorescence microscopy,

dot blotting and Western blotting, we found that SAL dramatically reduced the TRF2

protein levels in A549 cells (Fig. 2A). However, the levels of trf2 mRNA did not

change before and after SAL treatment (Fig. 2B upper). The proteasome inhibitor

MG-132 also could not reverse the SAL-induced down-regulation of TRF2 protein,

although MG-132 apparently enhanced the level of hypoxia-induction factor 1α

(HIF-1α) that is known to be degraded via the proteasome pathway(Li et al., 2004)

(Fig. 2B lower). Therefore, such reduction in TRF2 protein was irrelevant to TRF2

transcription or to TRF2 degradation via the proteasome pathway (Fig. 2B). We

further detected the ratio of the telomeric DNA bound to TRF2 relative to the total

telomeric DNA using telomere-ChIP assays. SAL was shown to significantly

decrease the telomeric DNA bound to TRF2, from 83.6 ± 2.4 % to 48.7 ± 1 % (Fig.

2C), indicating that some TRF2 was released from telomeric DNA. The data above

suggest that SAL is likely to potentiate damage to telomeric and genomic DNA by

disrupting the protection mechanism based on TRF2 protein.

TRF2 attenuates SAL-induced DNA damage and telomere erosion. To clarify


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the involvement of TRF2 to SAL-elicited DNA damage events, we over-espressed

TRF2 and down-regulated TRF2 expression using its small interfering RNAs

(siRNAs). The myc-tagged full length trf2 constructs and the basic/myb domain

double-deficient trf2-bm constructs (van Steensel et al., 1998) were transfected into

A549 cells and over-expressed effectively (Fig. 3A). Comet assays (Fig. 3B) and

γ-H2AX detection (Fig. 3C) showed that the full length trf2 transfection produced

fewer DSBs and lower levels of γ-H2AX in A549 cells following the treatment with

SAL compared to the vector transfection. Moreover, the telomere shortening (Fig. 3D)

and cytotoxicity (Fig. 3E) induced by SAL were also rescued by the full length trf2

transfection. In contrast, the double-deficient trf2-bm transfection, which would exert

a dominant-negative-like action (Smogorzewska and de Lange, 2002; van Steensel et

al., 1998), enhanced the genotoxic effects of SAL (Figs. 3B~E). These data show that

over-expression of TRF2 can protect cells from SAL-induced DNA damage and

telomere erosion.

On the other hand, we detected the effects of three TRF2 siRNAs with different

sequences in A549 cells. All the target sequecnces are located in the 3’-UTR of trf2

gene. Among them, the #2 TRF2 siRNA elicited the most prominent down-regulation

of TRF2 expression 24 h post transfection (Fig. 3F) and was chosen for the

subsequent experiments. The TRF2 siRNA transfection resulted in higher levels of

γ-H2AX (Fig. 3G) and more severe erosion of telomeres (Fig. 3H) induced by SAL

than the mock siRNA transfection. Our results indicate that the reduction of TRF2


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potentiates DNA DSBs and telomere shortening in response to genotoxic stress.

Taken together, our data from TRF2 over-expression and down-regulation

experiments suggest that TRF2 protein provides cells abilites to prevent telomeric

and non-telomeric DNA from genotoxic insults with a protection mechanism.

The DNA damage sensor ATR is required for the cellular response to the

SAL-induced DNA damage and telomere shortening. TRF2 has been suggested to

be coupled with DNA damage response (Bradshaw et al., 2005; Tanaka et al., 2005).

The protein kinases ATM and ATR are two key sensors for DNA damage signaling

(McGowan and Russell, 2004). To investigate how TRF2 protein is involved in DNA

damage events following SAL exposure, we firstly detected whether ATM and ATR

function in the SAL-induced DNA damage. Caffeine, an inhibitor for both ATM and

ATR (Blasina et al., 1999; Sarkaria et al., 1999; Zhou et al., 2000), nearly eliminated

SAL-induced γ-H2AX (Fig. 4A) and completely rescued telomeres from

SAL-induced erosion (Fig. 4D). However, only the down-regulation of ATR with

siRNA totally prevented the SAL-induced H2AX phosphorylation and telomere

erosion in A549 cells (Fig. 4C and 4E); ATM siRNA only reduced the levels of

γ-H2AX and marginally eased telomere shortening in the SAL-treated cells (Fig. 4B

and 4E). The data suggest that ATR is likely to play a more crucial role than ATM in

the cellular response to SAL-elicited DNA damage, especially to SAL-induced

telomere-erosion. However, both kinases may be required for the cellular response to

SAL-elicited DNA damage.


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TRF2 knockdown potentiates ATR activation, and ATR knockdown

prevents the TRF2 reduction following the treatment with SAL. The data above

show that TRF2 reduction is involved in DNA damage events induced by SAL and

that ATR is a critical factor for both DSBs and telomeric erosion elicited by SAL. In

order to investigate how TRF2 protein is involved in DNA damage events, we studied

the relationship between TRF2 and ATR. Firstly, we examined the change of ATR

activity following TRF2 knockdown. In the mock siRNA group, SAL enhanced the

ATR activity by about 1.0 fold. Surprisingly, TRF2 knockdown by siRNA led to an

additional 0.4 fold increment in the ATR activity in response to the SAL treatment

(Fig. 5A). We then investigated the effect of ATR knockdown on TRF2 loss induced

by SAL. The immunofluoresce of TRF2 showed that ATR knockdown by siRNA

unexpectedly rescued the loss of TRF2 protein resulted from the treatment with SAL

(Fig. 5B). These data suggest there might be a close relationship of mutual regulation

between the protein TRF2 and the kinase ATR, which is worthy of being studied

further.


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Discussion

We previously reported that SAL elicited DNA DSBs (Lu et al., 2005a; Meng and

Ding, 2001; Meng et al., 2001a; Meng et al., 2001b) and telomere erosion (Liu et al.,

2004) in separate systems. In the present study, using a single system in which SAL

did not induce early apoptosis (data not shown), we found that there exists a common

cellular response mechanism to both DNA DSBs and telomere erosion in

SAL-initiated events, i.e., SAL concurrently elicited both genomic DNA DSBs and

telomere erosion in A549 cells. Further studies highlight that this dual action, driven

by SAL , is in a TRF2-involved and ATR-mediated manner.

In tumor cells, the telomere length depends on the activity of telomerase, which

functions mainly in S phase during DNA replication. If the telomerase activity was

inhibited, telomere would shorten in the subsequent cell cycles, which would be a

long-term event. The other reason for telomere shortening is breakage in telomere

DNA, which can result from oxidative damage (von Zglinicki et al., 2000). Although

SAL inhibited telomerase activity after short-term treatment, it is obviously not the

main factor to shorten telomere within 3 h (Liu et al., 2004). Moreover, the

co-localization of DNA damage signals and telomere signals showed that the DNA

damage signals at the sites of telomeres increased with the concentrations of SAL

(Fig 1C), suggesting that the main reason for the rapid telomeric DNA loss induced

by SAL could result from the DNA breakage inside telomere structure. Any telomere

dysfunction resulting from mutation of telomere binding proteins, telomerase


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inhibition and destruction of telomere structure, would induce end-to-end fusion of

chromosomes in tumor cells. Abnormal chromosomes due to telomere dysfunction

would be harmful to the whole genome and activate DNA damage checkpoints, which

would activate apoptosis and inhibit cellular proliferation (Farazi et al., 2006; Herbig

and Sedivy, 2006; Lechel et al., 2005). Therefore, telomere erosion elicited by SAL in

short-time might be one important reason for the final fate of tumor cells , such as

proliferation inhibition and apopotosis under SAL treatment(Liu et al., 2004; Lu et al.,

2005a; Qing et al., 1999).

TRF2 is an important telomere binding protein; its deletion or mutation leads to

the loss of the telomeric 3'-tail and fusion of chromosome ends (Klapper et al., 2003;

Lechel et al., 2005; Li et al., 2005; Smogorzewska and de Lange, 2002; van Steensel

et al., 1998; Wang et al., 2004). TRF2 over-expression attenuates the ionizing

irradiation-initiated DNA damage response to DSBs and inhibits the ionizing

irradiation-induced cell cycle arrest in primary human fibroblasts (Bradshaw et al.,

2005; Karlseder et al., 2004). In our study, by either over-expression of wild-type or

mutated TRF2 or knockdown of TRF2 with its specific siRNAs, we demonstrate that

TRF2 protects telomeric and non-telomeric DNA from SAL-induced damage (Fig. 3).

In addition to its protective impact on telomere structure (Griffith et al., 1999;

Yoshimura et al., 2004), TRF2 is also implicated in DNA damage and DNA repair

systems (Bradshaw et al., 2005; Karlseder et al., 2004; Muftuoglu et al., 2006; Tanaka

et al., 2005; Zhu et al., 2000). In fact, TRF2 was capable of being translocated to


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ionizing irradiation-produced non-telomeric DSB sites and being phosphorylated in

an ATM-dependent manner (Bradshaw et al., 2005; Tanaka et al., 2005). TRF2 has

been shown to interact with the MRE11/RAD50/NRE repair complex and to

stimulate the activity of the repair enzyme DNA polymerase β (Muftuoglu et al., 2006;

Zhu et al., 2000). Moreover, it has been reported that TRF2 is required for repair of

non-telomeric DNA DSBs by homologous recombination (Mao et al., 2007). As such,

we propose that the SAL-elicited TRF2 disruption might deprive the cells of

protection, favoring shortening of telomere, and thus aggravation of DNA damage.

The protein kinases ATM and ATR are critical for DNA damage response

(McGowan and Russell, 2004). Their activation depends on the type of DNA damage.

Irradiation (IR) or other extracellular stimuli which elicit DSBs usually activate ATM

kinase(Shiloh, 2003). ATR has been shown usually activated by replication protein A

(RPA)-binding single-strand(Zou and Elledge, 2003). However, our studies show that

not only ATM but also ATR were involved in SAL-induced DNA DSBs (Fig.4), as

demonstrated that ATR could also respond to DSBs. Futhermore, our data is

consistent with the recent report that both ATM and ATR are activated in response to

IR-induced DSBs or UV-elicited DNA SSBs. And they activate different downstream

substrates in the DNA damage response systems(Helt et al., 2005). Therefore, the

involvement of ATR in SAL-induced DSBs might be explained that ATR

phosphorylates certain substrates that cooperate with ATM in response to DSBs

resulting from SAL.


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Given the fact that ATM and ATR are critical for DNA damage response

(McGowan and Russell, 2004), and ATM has been shown to mediate TRF2

phosphorylation (Tanaka et al., 2005) , which in turn inhibits ATM activity in a direct

manner (Celli and de Lange, 2005; Karlseder et al., 2004), we investigated the

possibility of a mutual regulation of both ATM and ATR in TRF2-driven events. By

using siRNA tools, we unexpectedly observed only ATR but not ATM is required for

SAL-elicited telomere erosion (Fig 4). Although further studies are needed with ATM

or ATR deficient cell lines, these data partially suggested an alternative view where

the activation of ATR and ATM might play an unequal role, at least in part, in

SAL-induced telomeric damage. The reason that ATM lacks its active commitment to

the TRF2-mediated telomeric DNA damage in response to SAL remains unclear,

which also deserves further investigation.

In addition, our findings that down-regulation of TRF2 by its specific siRNA led

to the enhanced activation of ATR induced by SAL, together with the fact that the

ATR siRNA transfection prevented the reduction of TRF2 in response to SAL

treatment, suggest a mutual negative feedback between the active ATR and TRF2 in

tumor development. We propose there are two possible mechanisms for the

interaction between ATR and TRF2. The first is that TRF2 might inhibit ATM

activation (Denchi and de Lange, 2007; Karlseder et al., 2004) as ATM could regulate

upstream ATR activation (Yoo et al., 2007); consequently, TRF2 indirectly induces

ATR repression. The other possible mechanism is that ATR might regulate the


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expression of TRF2 protein, suggested to be mediated by certain crosstalk between

their signal pathways. However, the detailed interaction between TRF2 and ATR still

needs to be investigated. This will go beyond the significance of SAL in cancer

therapy.

In summary, SAL concurrently induces telomeric and non-telomeric DNA

damage in A549 cells. The DNA damage activates the sensor kinase ATR, followed

by disruption of TRF2, which in turn negatively regulates ATR activation in response

to SAL. As a consequence, TRF2 reduction allows the genomic DNA and telomeres

to avoid the protection related to TRF2, thus potentiating the genotoxicity and

anticancer activity of SAL. This study provides the first line of evidence of TRF2

disruption in sensing DNA damage, and suggests the potential mutual regulation

between ATR and TRF2. With further research, it is likely that a connection between

telomere maintenance and DNA damage response in cancer development will be

elucidated.

Acknowledgements

We thank Professor de Lange Titia (Rockfeller University, USA) for the kind gift of

the TRF2 expression plasmids. We thank Dr. Andrew Jobson (National Cancer

Institute, USA) for the kind suggestion of the English writing.


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Footnotes:

This work was supported by the grants from the National Natural Sciences

Foundation of China (No. 30500618 and 30330670) and the Chinese Academy of

Sciences (the special scientific research initiation fund for Ze-Hong Miao, the winner

of the Special Prize of the President Scholarship).

The name and full address of person to receive reprint requests�Jian Ding, Ph.D. &

M.D., Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong

Zhi Road, Shanghai 201203, P. R. China. Tel +86-21-50806600, ext 2423; Fax

+86-21-50806722; E-mail: [email protected]


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Figure Legends

Figure 1. SAL concurrently induces DNA double-strand breaks and telomeric

erosion. A. A549 cells were treated with 25, 50 µM SAL or 100 µM VP16 for 3 h and

were harvested for detection of DNA double-strand breaks (DSBs) by neutral comet

assays. Representative “comet” images (200×) (left) from 3 separate experiments

were shown in which DNA was stained with DAPI (blue). Results were

semi-quantitated with the Komet 5.5 software and expressed as the Olive tail moment

(right). B. Phosphorylation of the histone H2AX (γ-H2AX) in the SAL-treated A549

cells. left: the levels of γ-H2AX protein detected by Western blotting; right:

Formation of γ-H2AX foci (green) examined by immunofluorescence confocal

microscopy. Nuclear outlines were superimposed using the respective DAPI images.

C. The damage sites on telomeric DNA. A549 cells were treated with 25, 50 µM SAL

for 3 h. Telomere signals were detected with a Cy3-labeled telomere PNA probe (red);

DNA breaks were presented with FITC-labeled dUTP (green).The DNA break sites

colocalized with the telomeres after SAL treatment (right), bar = 5 µm. D. Telomere

shortening was induced by SAL. Telomere length was detected by Flow-FISH with a

fluorescein-conjugated PNA probe. Representative histograms of flow cytometry

from 3 independent experiments (left) were revealed. Relative telomere length was

expressed as mean ± SD (right). Relative telomere length was the ratio of the

telomere fluorescence intensity in treated cells to that in the control; the telomere

fluorescence intensity = (Mean FL1 with probe – mean FL1 without probe). The


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fluorescence intensity of the control was taken as 100%.

Figure 2. SAL disrupts TRF2 protein in A549 cells. A. Reduction of TRF2 protein

by SAL. A549 cells were exposed to 50 µM SAL for 3 h and then subjected to

immunofluorescence confocal microscopy (left, bar=4 µm), dot blotting (middle) and

Western blotting (right). GAPDH was used as the loading control. Quantification for

the Western blotting data was done with Adobe Photoshop 7.0.1. B. SAL-induced

reduction of TRF2 protein was independent of the expression of TRF2 mRNA (upper)

and the degradation of TRF2 protein via the proteasome pathway (lower). The levels

of TRF2 mRNA were analyzed by RT-PCR. To detect the degradation of TRF2

protein via the proteasome pathway, A549 cells were treated with 50 µM SAL for 3 h

following pre-exposure to 10 µM MG-132 for 1 h , then subjected to Western blotting

to detect TRF2 (lower left) and Hif 1α (lower-right) protein level. C. Reduction of the

telomeric DNA bound to by TRF2. Telomere-CHIP assays were performed using a

TRF2 antibody to examine the telomeric DNA bound to by TRF2. upper: Telomeric

DNA signals were detected with the DIG-labeled telomere probe from the Roche

TeloTAGGG telomere detection system. Input: Supernatant before

immunoprecipitation; ppt: Protein-DNA immuno- precipitate complex. Normal

mouse IgG was used as the negative control. Lower: Telomere signals were

semi-quantitated with InGenius Bio-Imaging System. Telomeric DNA in CHIP (%) =

Telomeric DNA signals of ppt / Telomeric DNA signals of input ×100%.


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Figure 3. TRF2 attenuates SAL-induced DNA damage and telomere erosion. A.

Over-expression of TRF2. Upper: Schematic diagrams of human TRF2 and the

predicted products of different Myc-tagged trf2 alleles. Lower: Myc-tagged TRF2

proteins detected by Western blotting in total cell extracts from the monoclonal A549

cell population surviving over G418 selection post transfection. B. TRF2

over-expression decreased the SAL-induced DSBs examined by neutral gel

electrophoresis assays. ( # p<0.01 vs Vector without SAL group, **p<0.01 vs Vector

with SAL group). C. Reduced generation of γ-H2AX by SAL in TRF2 over-expressed

cells examined by Western blotting (left) and by confocal microscopy (right). D.

TRF2 over-expression prevented SAL-elicited telomere shortening. Telomere

fluorescent signals of the groups without SAL treatment were taken as 100%.

Relative telomere length of the corresponding SAL-treated groups was calculated as

the ratio of their fluorescence to that of their counterpart. The results were expressed

as means ± SD, n=3 (*p<0.05). E. TRF2 over-expression protected cells from

SAL’s cytotoxity measured by SRB assays. F. TRF2 knockdown 24 h post

transfection with 200 nM of siRNAs with different sequences. The relative density of

bands was analyzed with InGenius Bio-Imaging System. G. TRF2 down-regulation

enhanced the production of γ-H2AX by the SAL treatment. A549 cells were

transfected with 200 nM siRNA for 24 h followed by the exposure to 50 µM SAL or

100 µM VP16 for 3 h. Western blotting was done to test the levels of γ-H2AX. H.


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TRF2 knockdown aggravated the telomere shortening elicited by SAL. Telomere

fluorescence signals in the mock siRNA group without SAL were taken as 100%.

Figure 4. ATR is required for the cellular response to the SAL-induced DNA

damage and telomere shortening. A. Caffeine prevented the phosphorylation of the

histone H2AX. A549 cells were treated with 50 µM SAL for 3 h following the

pretreatment with 2 mM caffeine for 1 h and subjected to Western blotting (left) and

to immunofluorescence confocal microscopy (right). B. ATM siRNA alleviated the

phosphorylation of the histone H2AX by SAL. A549 cells were transfected with 100

nM ATM siRNA for 24 h. The cells were analyzed directly for ATM expression (left)

or continued to be exposed to SAL for additional 3 h for γ-H2AX detection (right) by

Western blotting. C. ATR knockdown blocked SAL-induced γ-H2AX production.

After 100 nM ATR siRNA transfection for 24 h, A549 cells were analyzed directly for

ATR expression (left) or continued to be exposed to SAL for an additional 3 h for

γ-H2AX detection (right) by Western blotting. D. Caffeine blocked the telomere

shortening induced by SAL. A549 cells were treated with 50 µM SAL for 3 h

following the pretreatment with 2 mM caffeine for 1 h and subjected to the relative

telomere length detection as in Fig. 1D. The telomere length of the control was taken

as 100%. The results were expressed as mean ± SD, n=3 (** p<0.01 vs the SAL alone

treated group). E. ATR was involved in SAL-induced telomeric length shortening.

Following ATM siRNA or ATR siRNA transfection as in B and C, A549 cells were


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exposed to 50 µM SAL for an additional 3 h. The relative telomere length was

analyzed and expressed as in D (** p<0.01 vs the SAL alone treated group).

Figure 5. TRF2 knockdown potentiates ATR activation and ATR knockdown

prevents TRF2 reduction following the treatment of SAL. A. A549 cells were

transfected with 200 nM TRF2 siRNA for 24 h and then treated with 50 µM SAL for an

additional 3 h. The cells were collected for ATR immunoprecipitation and in vitro ATR

activity assays. Representative images of the phospho-p53 substrate detected by

Western blotting were shown. Amount of phospho-p53 was semi-quantitated (up). B.

After 24 h transfection with 100 nM ATR siRNA, A549 cells were treated with 50 µM

SAL for an additional 3 h and then subjected to immunofluorescence microscopy for

TRF2 protein (green, left). The nuclei were stained with DAPI (blue), bar=4 µm.

Fluorescence of TRF2 protein was semi-quantitated by Photoshop 7.0 (right)


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The telomeric protein TRF2 is critical for the protection